Recombinant tumor specific antibody and use thereof

ABSTRACT

The invention provides a family of antibodies that specifically bind the human epithelial cell adhesion molecule. The antibodies comprise modified variable regions, more specially, modified framework regions, which reduce their immunogenicity when administered to a human. The antibodies, when coupled to the appropriate moiety, may be used in the diagnosis, prognosis and treatment of cancer.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Ser. No.60/288,564, filed May 3, 2001, the disclosure of which is incorporatedby reference herein.

FIELD OF THE INVENTION

The invention relates generally to recombinant antibodies. Moreparticulary, the invention relates to recombinant antibodies thatspecifically bind human Epithelial Cell Adhesion Molecule, and to theiruse as diagnostic, prognostic and therapeutic agents.

BACKGROUND OF THE INVENTION

There has been significant progress in the development of antibody-basedtherapies over the years. For example, investigators have identified notonly a variety of cancer-specific markers but also a variety ofantibodies that bind specifically to those markers. Antibodies can beused to deliver certain molecules, for example, a toxin or an immunestimulatory moiety, for example, a cytokine, to a cancer cell expressingthe marker so as to selectively kill the cancer cell (see, e.g., U.S.Pat. Nos. 5,541,087; and 5,650,150).

The KS-1/4 antibody is a mouse-derived monoclonal antibody directedagainst human epithelial cell adhesion molecule (EpCAM). EpCAM isexpressed at very low levels on the apical surface of certain epithelialcells. For example, EpCAM is expressed on intestinal cells on the cellsurface facing toward ingested food and away from the circulation, whereit would not be accessible to most proteins and cells of the immunesystem (Balzar et al. [1999] J. Mol. Med. 77:699-712).

Under certain circumstances, however, EpCAM is highly expressed oncertain cells, for example, tumor cells of epithelial origin. Typically,these tumor cells have lose their polarity with the result that EpCAM isexpressed over the entire surface of the cell. Thus, EpCAM is aconvenient tumor-specific marker for directing antibody-basedimmune-stimulatory moieties to tumor cells (Simon et al. [1990] Proc.Nat. Acad. Sci. USA 78:2755-2759; Perez et al. [1989] 7 Immunol.142:3662-3667).

However, antibodies can have an associated immunogenicity in the hostmammal. This is more likely to occur when the antibodies are notautologous. Consequently, the effectiveness of antibody-based therapiesoften is by an immunogenic response directed against the antibody. Theimmunogenic response typically is increased when the antibody is derivedin whole or in part from a mammal different than the host mammal, e.g.,when the antibody is derived from a mouse and the recipient is a human.Accordingly, it may be helpful to modify mouse-derived antibodies tomore closely resemble human antibodies, so as to reduce or minimize theimmunogenicity of the mouse-derived antibody.

Although a variety of approaches have been developed, including, forexample, chimeric antibodies, antibody humanization and antibodyveneering, Accordingly, there is a need in the art for antibodies thatbind to cancer specific markers and that have reduced immunogenicitywhen administered to a human. Further, there is a need in the art forantibodies that deliver toxins or immune stimulatory moieties, forexample, as fusion proteins or immune conjugates to a cancer specificmarker to selectively kill the tumor cell.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the identification ofrecombinant antibodies that specifically bind human EpCAM but are lessimmunogenic in humans than the template, murine anti-EpCAM antibodies.In particular, the invention provides recombinant KS antibodies in whichthe amino acid sequences defining one or more framework regions and/orcomplementarity determining regions have been modified to reduce theirimmunogenicity in humans.

As used herein, the terms “antibody” and “immunoglobulin” are understoodto mean (i) an intact antibody (for example, a monoclonal antibody orpolyclonal antibody), (ii) antigen binding portions thereof, including,for example, an Fab fragment, an Fab′ fragment, an (Fab′)₂ fragment, anFv fragment, a single chain antibody binding site, an sFv, (iii)bi-specific antibodies and antigen binding portions thereof, and (iv)multi, specific antibodies and antigen binding portions thereof.

As used herein, the terms “bind specifically,” “specifically bind” and“specific binding” are understood to mean that the antibody has abinding affinity for a particular antigen of at least about 10⁶ M⁻¹,more preferably, at least about 10⁷M⁻¹, more preferably at least about10⁸ M⁻¹, and most preferably at least about 10¹⁰M⁻¹.

As used herein, the terms “Complementarity-Determining Regions” and“CDRs” are understood to mean the hypervariable regions or loops of animmunoglobulin variable region that interact primarily with an antigen.The immunoglobulin heavy chain variable region (V_(H)) andimmunoglobulin light chain variable region (V_(L)) both contain threeCDRs interposed between framework regions, as shown in FIG. 1. Forexample, with reference to the amino acid sequence defining theimmunoglobulin light chain variable of the of the KS-1/4 antibody asshown in SEQ ID NO: 1, the CDRs are defined by the amino acid sequencesfrom Ser24 to Leu33 (CDR1), from Asp49 to Ser55 (CDR2), and from His88to Thr96 (CDR3). With reference to the amino acid sequence defining theimmunoglobulin heavy chain variable region of the KS-1/4 antibody asshown in SEQ ID NO: 2, the CDRs are defined by the amino acid sequencesfrom Gly26 to Asn35 (CDR1), from Trp50 to Gly66 (CDR2), and from Phe99to Tyr105 (CDR3). The corresponding CDRs of the other antibodiesdescribed herein are shown in FIGS. 1A-1C after alignment with thecorresponding KS-1/4 heavy or light chain sequence.

As used herein, the terms “Framework Regions” and “FRs” are understoodto mean the regions an immunoglobulin variable region adjacent to theComplementarity-Determining Regions. The immunoglobulin heavy chainvariable region (V_(H)) and immunoglobulin light chain variable region(V_(L)) both contain four FRs, as shown in FIG. 1. For example, withreference to the amino acid sequence defining the immunoglobulin lightchain variable of the of the KS-1/4 antibody as shown in SEQ ID NO: 1,the FRs are defined by the amino acid sequences from Gln1 to Cys23(FR1), from Trp34 to Phe 48 (FR2), from Gly56 to Cys87 (FR3), and fromPhe97 to Lys106 (FR4). With reference to the amino acid sequencedefining the immunoglobulin heavy chain variable region of the KS-1/4antibody as shown in SEQ ID NO: 2, the FRs are defined by the amino acidsequences from Gln1 to Ser25 (FR1), from Trp36 to Gly49 (FR2), fromArg67 to Arg98 (FR3), and from Trp106 to Ser116 (FR4). The FRs of theother antibodies described herein are shown in Figures X and Y afteralignment with the corresponding KS-1/4 heavy or light chain sequence.

As used herein, the term “KS antibody” is understood to mean an antibodythat binds specifically to the same human EpCAM antigen bound by murineantibody KS-1/4 expressed by a hybridoma (see, for example, Cancer Res.1984, 44 ((2):681-7). The KS antibody preferably comprises (i) an aminoacid sequence of SASSSVSY (amino acids 24-31 of SEQ ID NO: 1) definingat least a portion of an immunoglobulin light chain CDR1 sequence, (ii)an amino acid sequence of DTSNLAS (amino acids 49-55 of SEQ ID NO: 1)defining at least a portion of an immunoglobulin light chain CDR2sequence, (iii) an amino acid sequence of HQRSGYPYT (amino acids 88-96of SEQ ID NO: 1) defining at least a portion of an immunoglobulin lightchain CDR3 sequence, (iv) an amino acid sequence of GYTFTNYGMN (aminoacids 26-35 of SEQ ID NO: 2) defining at least a portion of animmunoglobulin heavy chain CDR1 sequence, (v) an amino acid sequence ofWINTYTGEPTYAD (amino acids 50-62 of SEQ ID NO: 2) defining at least aportion of an immunoglobulin heavy chain CDR2 sequence, or (vi) an aminoacid sequence of SKGDY (amino acids 101-105 of SEQ ID NO: 2) defining atleast a portion of an immunoglobulin heavy chain CDR3 sequence, or anycombination of the foregoing.

In one aspect, the invention provides a recombinant antibody thatspecifically binds EpCAM, wherein the antibody comprises an amino acidsequence, a portion of which defines a framework region in animmunoglobulin V_(L) domain. In one embodiment, the framework region(FR1) is defined by amino acid residues 1-23 of SEQ ID NO: 5, whereinXaa1 is Q or E, Xaa3 is L or V, Xaa10 is I or T, Xaa11 is M or Xaa13 isA or L, Xaa18 is K or R, or Xaa21 is M or L, provided that at least oneof the amino acid residues at positions Xaa1, Xaa3, Xaa10, Xaa11, Xaa13,Xaa18, or Xaa21 is not the same as the amino acid at the correspondingposition in SEQ ID NO: 1. The amino acids at each of the positions aredenoted by the standard single letter code.

In another embodiment, the framework region (FR2) is defined by aminoacid residues 34-48 of SEQ ID NO: 5, wherein Xaa41 is S or Q, Xaa42 is Sor A, Xaa45 is P or L, or Xaa46 is W or L, provided that at least one ofthe amino acid residues at positions Xaa41, Xaa42, Xaa45, or Xaa46 isnot the same as the amino acid at the corresponding position in SEQ IDNO: 1.

In another embodiment, the framework region (FR3) is defined by aminoacid residues 56-87 of SEQ ID NO: 5, wherein Xaa57 is F or I, Xaa69 is Sor D, Xaa71 is S or T, Xaa73 is I or T, Xaa77 is M or L, Xaa79 is A orP, Xaa82 is A or F, or Xaa84 is T or V, provided that at least one ofthe amino acid residues at positions Xaa57, Xaa69, Xaa71, Xaa73, Xaa77,Xaa79, Xaa82, or Xaa84 is not the same as the amino acid at thecorresponding position in SEQ ID NO: 1.

In another aspect, the invention provides a recombinant antibody thatspecifically binds EpCAM, wherein the antibody comprises an amino acidsequence, a portion of which defines a framework region in animmunoglobulin V_(I), domain. In one embodiment, the framework region(FR1) is defined by amino acid residues 1-25 of SEQ ID NO: 6, whereinXaa2 is I or V, Xaa9 is P or A, Xaa11 is L or V, or Xaa17 is T or S,provided that at least one of the amino acid residues at positions Xaa2,Xaa9, Xaa11 or Xaa17 is not the same as the amino acid at thecorresponding position in SEQ ID NO: 2.

In another embodiment, the framework region (FR2) is defined by aminoacid residues 36-49 of SEQ ID NO: 6, wherein Xaa38 is K or R, Xaa40 is Tor A, or Xaa46 is K or E, provided that at least one of the amino acidresidues at positions Xaa38, Xaa40, Xaa46 is not the same as the aminoacid at the corresponding position in SEQ ID NO: 2.

In another embodiment, the framework region (FR3) is defined by aminoacid residues 67-98 of SEQ ID NO: 6, wherein Xaa68 is F or V, Xaa69 is Aor T, Xaa70 is F or I, Xaa73 is E or D, Xaa76 is A or T, Xaa80 is F orY, Xaa83 is I or L, Xaa84 is N or S, Xaa85 is N or S, Xaa88 is N, A orS, Xaa91 is M or T, or Xaa93 is T or V, provided that at least one ofthe amino acid residues at positions Xaa68, Xaa69, Xaa70, Xaa73, Xaa76,Xaa80, Xaa83, Xaa84, Xaa85, Xaa88, Xaa91 or Xaa93 is not the same as theamino acid at the corresponding position in SEQ ID NO: 2. In anotherembodiment, the framework region (FR4) is defined by amino acid residues106-116 of SEQ ID NO: 6, wherein Xaa108 is Q or T.

In another embodiment, the immunoglobulin V_(L) domain comprises an FR1sequence selected from the group consisting of (i) amino acid residues1-23 of SEQ ID NO: 9; and (ii) amino acid residues 1-23 of SEQ ID NO: 8.In another embodiment, the immunoglobulin V_(H) domains comprises an FRsequence defined by amino acid residues 1-25 of SEQ ID NO: 18 and or anFR sequence defined by amino acid residues 67-98 of SEQ ID NO: 18. Morepreferably, the V_(L) domain comprises an amino acid sequence defined byamino acids 1-106 of SEQ ID NO: 9 and/or the V_(H) domain comprises anamino acid sequence defined by amino acids 1-116 of SEQ ID NO: 18.

Furthermore, the antibody optionally may include an amino acid sequencedefining at least a portion of a CDR sequence including, for example,(i) amino acid residues 24-31 of SEQ ID NO: 1; (ii) amino acid residues49-55 of SEQ ID NO: 1; and/or (iii) amino acid residues 88-96 of SEQ IDNO: 1. Similarly, the antibody optionally may include an amino acidsequence defining at least a portion of a CDR sequence including, forexample, (i) amino acid residues 26-35 of SEQ ID NO: 2; (ii) amino acidresidues 50-62 of SEQ ID NO: 2; and/or iii) amino acid residues 101-105of SEQ ID NO: 2.

In another embodiment, the antibody comprises the antigen targetingportion of an antibody-cytokine fusion protein. The cytokine preferablyis an interleukin and more preferably is interleukin-2.

In another aspect, the invention provides an expression vector encodingat least a portion of the antibody of the invention. In a preferredembodiment, the expression vector comprises the nucleotide sequence setforth in SEQ ID NO: 40.

In another aspect, the invention provides a method of diagnosing,prognosing and/or treating a human patient having a disease associatedwith over-expression of EpCAM (for example, a disease in which EpCAM ispresent at a higher level in diseased tissue relative to tissue withoutthat disease). The method comprises administering one of the antibodiesof the invention to an individual in need of such diagnosis, prognosisor treatment.

The antibody optionally includes a diagnostic and/or therapeutic agentattached thereto. The agent may be fused to the antibody to produce afusion protein. Alternatively, the agent may be chemically coupled tothe antibody to produce an immuno-conjugate. It is contemplated that theagent may include, for example, a toxin, radiolabel, cytokine, imagingagent or the like. In a preferred embodiment, the antibody of theinvention is fused as a fusion protein to a cytokine. Preferredcytokines preferably include interleukins such as interleukin-2 (IL-2),IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16 andIL-18, hematopoietic factors such as granulocyte-macrophage colonystimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF) and erythropoeitin, tumor necrosis factors (TNF) such as TNFα,lymphokines such as lymphotoxin, regulators of metabolic processes suchas leptin, interferons such as interferon α, interferon β, andinterferon γ, and chemokines. Preferably, the antibody-cytokine fusionprotein displays cytokine biological activity.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show an alignment of light and heavy chain variantsand consensus sequences of KS antibodies. The immunoglobulin FrameworkRegions (FR1-FR4) are denoted by-. The immunoglobulin ComplementarityDetermining Regions (CDR1-CDR3) are denoted by*. Individual KS antibodylight chain V region segments are referred to as “VK,” wherein K refersto the fact that the light chain is a kappa chain. Individual KSantibody heavy chain V region segments are referred to as “V_(H).”Substitutable amino acids are denoted by “X” in the consensus sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides recombinant antibodies that specificallybind human Epithelial Cell Adhesion Molecule (EpCAM). Preferredantibodies of the invention have altered variable regions that result inreduced immunogenicity in humans. Antibody variable regions of theinvention are particularly useful to target antibodies and antibodyfusion proteins to tumor tissues that over-express EpCAM in humanpatients. In preferred embodiments, an antibody of the invention isfused to a cytokine to produce an immuno-cytokine.

Protein Sequences of the Invention

The present invention discloses a family of antibody variable region orV region sequences that, when appropriately heterodimerized, bind tohuman epithelial cell adhesion molecule (EpCAM) also known as KS antigenor KSA. Preferred proteins of the invention are useful for treatinghuman patients as described herein. Accordingly, preferred KS antibodyvariants are humanized, deimmunized, or both, in order to reduce theirimmunogenicity when administered to a human. According to the invention,murine KS antibodies can be deimmunized or humanized, for example, byusing deimmunization methods in which potential T cell epitopes areeliminated or weakened by introduction of mutations that reduce bindingof a peptide epitope to an MHC Class II molecule (see, for exampleWO98/52976, and WO00/34317), or by using methods in which non-human Tcell epitopes are mutated so that they correspond to human self epitopesthat are present in human antibodies (see, for example, U.S. Pat. No.5,712,120).

I. Variable Light Chain

The recombinant anti-EpCAM antibody has an immunoglobulin variable lightchain sequence having the following amino acid sequence:

(SEQ ID NO: 3) X-I-X-L-T-Q-S-P-A-X-X-X-X-S-P-G-X-X-X-T-X-T-C-S-A-S-S-S-V-S-T-X-L-W-Y-X-Q-K-P-G-X-X-P-K-X-X-I-X-D-T-S-N-L-A-S-G-X-P-X-R-F-S-G-S-G-S-G-T-X-Y-X-L-X-I-X-S-X-E-X-E-D-X-A-X-Y-Y-C-H-Q-R-S-G-Y-P-Y-T-F-G- G-G-T-K-X-E-I-K.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin light chain FR1, which isrepresented by residues 1 to 23 of SEQ ID NO: 3, namely,X-I-X-L-T-Q-S-P-A-X-X-X-X-S-P-G-X-X-X-T-X-T-C. More particularly, therecombinant anti-EpCAM antibody has at least one of the following aminoacids in the FR1 region: Q or Eat position Xaa1; L or V at positionXaa3; I, T or S at position Xaa10; M or L at position Xaa11; S or A atposition Xaa12; A, L or V at position Xaa13; E or Q at position Xaa17, Kor R at position Xaa18, V or A at position Xaa19; and, M, L or I atposition Xaa21. More preferably, the recombinant anti-EpCAM antibody hasat least one of the following amino acid substitutions in the FR1region: E at position Xaa1; V at position Xaa3; T or S at positionXaa10; L at position Xaa11; A at position Xaa12; L or V at positionXaa13; Q at position Xaa17, R at position Xaa18, A at position Xaa19;and, L or I at position Xaa21.

In another embodiment, the recombinant anti-EpCAM antibody of theinvention has an amino acid sequence defining an immunoglobulin lightchain CDR1, which is represented by residues 24 to 33 of SEQ ID NO: 3,namely S-A-S-S-S-V-S-T-X-L. More particularly, the recombinantanti-EpCAM antibody of the invention has one of the following aminoacids in the CDR1 region: M or I at position Xaa32. More preferably, therecombinant anti-EpCAM antibody has an amino acid substitution in theCDR1 region, for example, I at position Xaa32.

In another embodiment, the recombinant anti-EpCAM antibody has an aminoacid sequence defining an immunoglobulin light chain FR2, which isrepresented by residues 34 to 48 of SEQ ID NO: 3, namelyW-Y-X-Q-K-P-G-X-X-P-K-X-X-I-X. More particularly, the recombinantanti-EpCAM antibody has at least one of the following amino acids in theFR2 region: Q or L at position Xaa36; S or Q at position Xaa41; S, A orP at position Xaa42; P or L at position Xaa45; W or L at position Xaa46;and, F or Y at position Xaa48. More preferably, the recombinantanti-EpCAM antibody has at least one of the following amino acidsubstitutions in the FR2 region: L at position Xaa36; Q at positionXaa41; A or P at position Xaa42; L at position Xaa45; L at positionXaa46; and, Y at position Xaa48.

In another embodiment, the recombinant anti-EpCAM antibody has an aminoacid sequence defining an immunoglobulin light chain FR3, which isrepresented by residues 56 to 87 of SEQ ID NO: 3, namely,G-X-P-X-R-F-S-G-S-G-S-G-T-X-Y-X-L-X-I-X-S-X-E-X-E-D-X-A-X-Y-Y-C. Moreparticularly, the recombinant anti-EpCAM antibody has at least one ofthe following amino acids in the FR3 region: F or I at position Xaa57; Aor S at position Xaa59; S, D or T at position Xaa69; I or T at positionXaa71; For T at position Xaa73; S or N at position Xaa75; M or L atposition Xaa77; A or P at position Xaa79; A or F at position Xaa82; and,T or V at position Xaa84. More preferably, the recombinant anti-EpCAMantibody has at least one of the following amino acid substitution inthe FR3 region: I at position Xaa57; S at position Xaa59; D or T atposition Xaa69; T at position Xaa71; T at position Xaa73; N at positionXaa75; L at position Xaa77; P at position Xaa79; F at position Xaa82;and, V at position Xaa84.

In another embodiment, the recombinant anti-EpCAM antibody has an aminoacid sequence defining an immunoglobulin light chain FR4, which isrepresented by residues 97 to 106 of SEQ ID NO: 3, namely,F-G-G-G-T-K-X-E-I-K. More particularly, the recombinant anti-EpCAMantibody of the invention has at least one of the following amino acidsin the FR4 region, for example, L or V at position Xaa103. Accordingly,the recombinant anti-EpCAM antibody of the invention has an amino acidsubstitution in the FR4 region, for example, V at position Xaa103.

II. Variable Heavy Chain

The recombinant anti-EpCAM antibody has an immunoglobulin variable heavychain sequence having the following amino acid sequence:

(SEQ ID NO: 4) Q-X-Q-L-V-Q-S-G-X-E-X-K-K-P-G-X-X-V-K-I-S-C-K-A-S-G-Y-T-F-T-N-Y-G-M-N-W-V-X-Q-X-P-G-X-G-L-X-W-M-G-W-I-N-T-Y-T-G-E-P-T-Y-A-D-X-F-X-G-R-X-X-X-X-X-X-T-S-X-S-T-X-X-L-Q-X-X-X-L-R-X-E-D-X-A-X-Y-F-C-V-R-F-X-S-K-G-D-Y-W-G-X-G-T-X-V-T-V-S-S

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR1, which isrepresented by residues 1 to 25 of SEQ ID NO: 4, namelyQ-X-Q-L-V-Q-S-G-X-E-X-K-K-P-G-X-X-V-K-I-S-C-K-A-S. More particularly,the recombinant anti-EpCAM antibody has at least one of the followingamino acids in the FR1 region: I or V at position Xaa2; P or A atposition Xaa9; L or V at position Xaa11; E or S at position Xaa16; and,T or S at position Xaa17. More preferably, the recombinant anti-EpCAMantibody has at least one of the following amino acid substitutions inthe FR1 region: V at position Xaa2; A at position. Xaa9; V at positionXaa11; S at position Xaa16; and, S at position Xaa17.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR2, which isrepresented by residues 36 to 49 of SEQ ID NO: 4,W-V-X-Q-X-P-G-X-G-L-X-W-M-G. More particularly, the recombinantanti-EpCAM antibody has at least one of the following amino acids in theFR2 region: K or R at position Xaa38; T or A at position Xaa40; K or Qat position Xaa43; and, K or E at position Xaa46. More preferably, therecombinant anti-EpCAM antibody has at least one of the following aminoacid substitutions in the FR2 region: R at position Xaa38; A at positionXaa40; Q at position Xaa43; and, E at position Xaa46.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain CDR2, whichis represented by residues 50 to 66 of SEQ ID NO: 4, namelyW-I-N-T-Y-T-G-E-P-T-Y-A-D-X-F-X-G. More particularly, the recombinantanti-EpCAM antibody has at least one of the following amino acids in theCDR2 region: D or K at position Xaa63; and, K or Q at position Xaa65.More preferably, the recombinant anti-EpCAM antibody has at least one ofthe following amino acid substitutions in the CDR2 region: K at positionXaa63; and, Q at position Xaa65.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR3, which isrepresented by residues 67 to 98 of SEQ ID NO: 4, namelyR-X-X-X-X-X-X-T-S-X-S-T-X-X-L-Q-X-X-X-L-R-X-E-D-X-A-X-Y-F-C-V-R. Moreparticularly, the recombinant anti-EpCAM antibody of the invention hasat least one of the following amino acids in the FR3 region: F or V atposition Xaa68, A, T or V at position Xaa69; F or I at position Xaa70; Sor T at position Xaa71; L or A at position Xaa72; E or D at positionXaa73; A or T at position Xaa76; A or L at position Xaa79; F or Y atposition Xaa80; I or L at position Xaa83; N or S at position Xaa84; N orS at position Xaa85; N, A or S at position Xaa88; M or T at positionXaa91; and, T or V at position Xaa93. More preferably, the recombinantanti-EpCAM antibody has at least one of the following amino acidsubstitutions in the FR3 region: V at position Xaa68, T or V at positionXaa69; I at position Xaa70; T at position Xaa71; A at position Xaa72; Dat position Xaa73; T at position Xaa76; L at position Xaa79; Y atposition Xaa80; L at position Xaa83; S at position Xaa84; S at positionXaa85; A or S at position Xaa88; T at position Xaa91; and, V at positionXaa93.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain CDR3, whichis represented by residues 99 to 105 of SEQ ID NO: 4, namelyF-X-S-K-G-D-Y. More particularly, the recombinant anti-EpCAM antibodyhas at least one of the following amino acids in the CDR3 region, forexample, I or M at position Xaa100. More preferably, the recombinantanti-EpCAM antibody has an amino acid substitution in the CDR3 region,for example, M at position Xaa100.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR4, which isrepresented by residues 106 to 116 of SEQ ID NO: 4, namelyW-G-X-G-T-X-V-T-V-S-S. More particularly, the recombinant anti-EpCAMantibody has at least one of the following amino acids in the FR4region: Q or T at position Xaa108; and, S or T at position X111. Morepreferably, the recombinant anti-EpCAM antibody has at least one of thefollowing amino acid substitutions in the FR4 region: T at positionXaa108; and, T at position X111.

III. Refined Variable Light Chain

In another embodiment, the recombinant anti-EpCAM antibody has animmunoglobulin variable light chain sequence having the following aminoacid sequence:

(SEQ ID NO: 5) X-I-X-L-T-Q-S-P-A-X-X-S-X-S-P-G-E-X-V-T-X-T-C-S-A-S-S-S-V-S-Y-M-L-W-Y-Q-Q-K-P-G-X-X-P-K-X-X-I-F-D-T-S-N-L-A-S-G-X-P-A-R-F-S-G-S-G-S-G-T-X-Y-X-L-X-I-S-S-X-E-X-E-D-X-A-X-Y-Y-C -H-Q-R-S-G-Y-P-Y-T-F-G-G- G-T-K-L-E-I-K

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin light chain FR1, which isrepresented by residues 1 to 23 of SEQ ID NO: 5, namelyX-I-X-L-T-Q-S-P-A-X-X-S-X-S-P-G-E-X-V-T-X-T-C. More particularly, therecombinant anti-EpCAM antibody has at least one of the following aminoacids in the FR1 region: Q or E at position Xaa1; L or V at positionXaa3; I or T at position Xaa10; M or L at position Xaa11; A or L atposition Xaa13; K or R at position Xaa18; and, M or L at position Xaa21.More preferably, the recombinant anti-EpCAM antibody has at least one ofthe following amino acid substitutions in the FR1 region: E at positionXaa1; V at position Xaa3; T at position Xaa10; L at position Xaa11; L atposition Xaa13; Rat position Xaa18; and, L at position Xaa21.

In another preferred embodiment, the recombinant anti-EpCAM antibody hasan amino acid sequence defining an immunoglobulin light FR1 having atleast one of the following amino acids in the FR1 region: Q or E atposition Xaa1; A or L at position Xaa11; and, M or L at position Xaa21.More preferably, the recombinant anti-EpCAM antibody has an amino acidsequence defining an immunoglobulin light FR1 having at least one of thefollowing substitutions in the FR1 region: E at position Xaa1; L atposition Xaa11; and, L at position Xaa21.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin light chain FR2, which isrepresented by residues 34 to 48 of SEQ ID NO: 5, namelyW-Y-Q-Q-K-P-G-X-X-P-K-X-X-I-F. More preferably, the recombinantanti-EpCAM antibody has at least one of the following amino acids in theFR2 region: S or Q at position Xaa41; S or A at position Xaa42; P or Lat position Xaa45; and, W or L at position Xaa46. More preferably, therecombinant anti-EpCAM antibody has at least one of the following aminoacid substitutions in the FR2 region: Q at position Xaa41; A at positionXaa42; L at position Xaa45; and, L at position Xaa46.

In another preferred embodiment, the recombinant anti-EpCAM antibody ofthe invention has an amino acid sequence defining an immunoglobulinlight FR2 having at least one of the following amino acids in the FR2region: S or A at position Xaa42; P or L at position Xaa45; and, W or Lat position Xaa46. More preferably, the recombinant. anti-EpCAM antibodyhas an amino acid sequence defining an immunoglobulin light FR2 havingat least one of the following substitutions in the FR2 region: A atposition Xaa42; L at position Xaa45; and, L at position Xaa46.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin light chain FR3, which isrepresented by residues 56 to 87 of SEQ ID NO: 5, namelyG-X-P-A-R-F-S-G-S-G-S-G-T-X-Y-X-L-X-I-S-S-X-E-X-E-D-X-A-X-Y-Y-C. Moreparticularly, the recombinant anti-EpCAM antibody has at least one ofthe following amino acids in the FR3 region: F or I at position Xaa57; Sor D at position Xaa69; S or T at position Xaa71; I or T at positionXaa73; M or L at position Xaa77; A or P at position Xaa79; A or F atposition Xaa82; and, T or V at position Xaa84. More preferably, therecombinant anti-EpCAM antibody has at least one of the following aminoacid substitution in the FR3 region: I at position Xaa57; D at positionXaa69; T at position Xaa71; T at position Xaa73; L at position Xaa77; Pat position Xaa79; F at position Xaa82; and, V at position Xaa84.

In another preferred embodiment, the recombinant anti-EpCAM antibody ofthe invention has an amino acid sequence defining an immunoglobulinlight FR3 having at least one of the following amino acids in the FR3region: F or I at position Xaa57; S or D at position Xaa69; A or P atposition Xaa79; A or F at position Xaa82; and, T or V at position Xaa84.More preferably, the recombinant anti-EpCAM antibody has an amino acidsequence defining an immunoglobulin light FR3 having at least one of thefollowing substitutions in the FR3 region: I at position Xaa57; D atposition Xaa69; P at position Xaa79; F at position Xaa82; and, V atposition Xaa84.

IV. Refilled Variable Heavy Chain

The recombinant anti-EpCAM antibody has an immunoglobulin variable heavychain sequence having the following amino acid sequence:

(SEQ ID NO: 6) Q-X-Q-L-V-Z-S-G-X-E-X-K-K-P-G-E-X-V-K-I-S-C-K-A-S-G-Y-T-F-T-N-Y-G-M-N-W-V-X-Q-X-P-G-K-G-L-X-W-M-G-W-I-N-T-Y-T-G-E-P-T-Y-A-D-X-F-X-G-R-X-X-X-S-L-X-T-S-X-S-T-A-X-L-Q-X-X-X-L-R-X-E-D-X-A-X-Y-F-C-V-R-F-I-S-K-G-D-Y-W-G-Q-G-T-S-V-T-V-S-S

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR1, which isrepresented by residues 1 to 25 of SEQ ID NO: 6, namelyQ-X-Q-L-V-Q-S-G-X-E-X-K-K-P-G-E-X-V-K-I-S-C-K-A-S. More preferably, therecombinant anti-EpCAM antibody has at least one of the following aminoacids in the FR1 region: I or V at position Xaa2; P or A at positionXaa9; L or V at position Xaa11; and, T or S at position Xaa17.Accordingly, a recombinant anti-EpCAM antibody of the invention has atleast one of the following amino acid substitution in the FR1 region: Vat position Xaa2; A at position Xaa9; V at position Xaa11; and, S atposition Xaa17.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy FR1 having at leastone of the following amino acids in the FR1 region: I or V at positionXaa2; P or A at position Xaa9; and, L or V at position Xaa11.Accordingly, a recombinant anti-EpCAM antibody of the invention has anamino acid sequence defining an immunoglobulin heavy FR1 having at leastone of the following substitutions in the FR1 region: V at positionXaa2; A at position Xaa9; and, V at position Xaa11.

In another embodiment, a recombinant anti-EpCAM antibody of theinvention has an amino acid sequence defining an immunoglobulin heavychain FR2, which is represented by residues 36 to 49 of SEQ ID NO: 6,namely W-V-X-Q-X-P-G-K-G-L-X-W-M-G. More particularly, the recombinantanti-EpCAM antibody has at least one of the following amino acidsubstitution in the FR2 region: K or R at position Xaa38; T or A atposition Xaa40; and, K or E at position Xaa46. More preferably, therecombinant anti-EpCAM antibody has at least one of the following aminoacid substitution in the FR2 region: R at position Xaa38; A at positionXaa40; and, E at position Xaa46.

In another preferred embodiment, the recombinant anti-EpCAM antibody hasan amino acid sequence defining an immunoglobulin heavy FR2 having thefollowing amino acids in the FR1 region, for example, K or E at positionXaa46. More preferably, the recombinant anti-EpCAM antibody has an aminoacid sequence defining an immunoglobulin heavy FR2 having an amino acidsubstitution in the FR1 region, for example, E at position Xaa46.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain CDR2, whichis represented by residues 50 to 66 of SEQ ID NO: 6, namelyW-I-N-T-Y-T-G-E-P-T-Y-A-D-X-F-X-G. More particularly, the recombinantanti-EpCAM antibody has at least one of the following amino acids in theCDR2 region: D or K at position Xaa63; and, K or Q at position Xaa65.More preferably, the recombinant anti-EpCAM antibody has at least one ofthe following amino acid substitutions in the CDR2 region: K at positionXaa63; and, Q at position Xaa65.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR3, which isrepresented by residues 67 to 98 of SEQ ID NO: 6, namelyR-X-X-X-S-L-X-T-S-X-S-T-A-X-L-Q-X-X-X-L-R-X-E-D-X-A-X-Y-F-C-V-R. Moreparticularly, the recombinant anti-EpCAM antibody of the invention hasat least one of the following amino acids in the FR3 region: F or V atposition Xaa68; A or T at position Xaa69; F or I at position Xaa70; E orD at position Xaa73; A or T at position Xaa76; F or Y at position Xaa80;I or L at position Xaa83; N or S at position Xaa84; N or S at positionXaa85; N, A or S at position Xaa88; M or T at position Xaa91; and, T orV at position Xaa93. More preferably, the recombinant anti-EpCAMantibody has at least one of the following amino acid substitutions inthe FR3 region: V at position Xaa68; T at position Xaa69; I at positionXaa70; D at position Xaa73; T at position Xaa76; Y at position Xaa80; Lat position Xaa83; S at position Xaa84; S at position Xaa85; A or S atposition Xaa88; T at position Xaa91; and, V at position Xaa93.

In another preferred embodiment, the recombinant anti-EpCAM antibody ofthe invention has an amino acid sequence defining an immunoglobulinheavy chain FR3 having at least one of the following amino acids in theFR3 region: F or V at position Xaa68; E or D at position Xaa73; N or Sat position Xaa84; N or S at position Xaa85; N or A at position Xaa88;and, T or V at position Xaa93. More preferrably, the recombinantanti-EpCAM antibody has an amino acid sequence defining animmunoglobulin heavy FR3 having at least one of the followingsubstitutions in the FR3 region: V at position Xaa68; D at positionXaa73; S at position Xaa84; S at position Xaa85; A at position Xaa88;and, V at position Xaa93.

In a preferred embodiment, the recombinant anti-EpCAM antibody has anamino acid sequence defining an immunoglobulin heavy chain FR4, which isrepresented by residues 106 to 116 of SEQ ID NO: 6, namelyW-G-X-G-T-S-V-T-V-S-S. More particularly, the recombinant anti-EpCAMantibody has at least one of the following amino acids in the FR4region, for example, Q or T at position Xaa108. More preferably, therecombinant anti-EpCAM antibody has an amino acid substitution in theFR4 region, for example, T at position Xaa108.

Accordingly, preferred V regions contain substitutions in FR domains ofV_(H) and/or VK regions corresponding to murine KS-1/4 variable regions.In addition, preferred V regions of the invention do not includeinsertions or deletions of amino acids relative to the murine KS-1/4variable regions;

Preferred variants include proteins having variable regions with greaterthan 80% identity/homology murine KS-1/4. The amino acid sequence ofmurine KS variable region or a portion thereof may be used as areference sequence to determine whether a candidate sequence possessessufficient amino acid similarity to have a reasonable expectation ofsuccess in the methods of the present invention. Preferably, variantsequences are at least 70% similar or 60% identical, more preferably atleast 75% similar or 65% identical, and most preferably 80% similar or70% identical to a murine KS variable heavy or light chain FR or CDR.

To determine whether a candidate peptide region has the requisitepercentage similarity or identity to a murine KS sequence, the candidateamino acid sequence and murine KS sequence are first aligned using thedynamic programming algorithm described in Smith and Waterman (1981) J.Mol. Biol. 147:195-197, in combination with the BLOSUM62 substitutionmatrix described in FIG. 2 of Henikoff and Henikoff (1992) PNAS89:10915-10919. For the present invention, an appropriate value for thegap insertion penalty is −12, and an appropriate value for the gapextension penalty is −4. Computer programs performing alignments usingthe algorithm of Smith-Waterman and the BLOSUM62 matrix, such as the GCGprogram suite (Oxford Molecular Group, Oxford, England), arecommercially available and widely used by those skilled in the art. Oncethe alignment between the candidate and reference sequence is made, apercent similarity score may be calculated. The individual amino acidsof each sequence are compared sequentially according to their similarityto each other. If the value in the BLOSUM62 matrix corresponding to thetwo aligned amino acids is zero or a negative number, the pairwisesimilarity score is zero; otherwise the pairwise similarity score is1.0. The raw similarity score is the sum of the pairwise similarityscores of the aligned amino acids. The raw score is then normalized bydividing it by the number of amino acids in the smaller of the candidateor reference sequences. The normalized raw score is the percentsimilarity. Alternatively, to calculate a percent identity, the alignedamino acids of each sequence are again compared sequentially. If theamino acids are non-identical, the pairwise identity score is zero;otherwise the pairwise identity score is 1.0. The raw identity score isthe sum of the identical aligned amino acids. The raw score is thennormalized by dividing it by the number of amino acids in the smaller ofthe candidate or reference sequences. The normalized raw score is thepercent identity. Insertions and deletions are ignored for the purposesof calculating percent similarity and identity. Accordingly, gappenalties are not used in this calculation, although they are used inthe initial alignment.

The invention also discloses methods for assaying the expression of KSantibodies from cells such as mammalian cells, insect cells, plantcells, yeast cells, other eukaryotic cells or prokaryotic cells (seeExample 1). In a preferred method, KS antibody V regions are expressedas components of an intact human antibody, and the expression of theantibody from a eukaryotic cell line assayed by an ELISA that detectsthe human Fc region. To precisely quantify binding of a KS antibody toEpCAM, a Biacore assay may be used.

Treatment of Human Disease with KS Antibody Fusion Proteins

The invention also discloses the sequences of KS antibody-IL2 fusionproteins that are useful in treating human disease, such as cancer.Certain KS antibody-IL2 fusion proteins, such as KS-1/4-IL2 (see, forexample, Construct 3 in Example X), may be used to treat human patientswith cancer, with surprisingly little immune response against theantibody.

It is found that, during treatment of human cancers withKS-1/4(VH2/VK1)-IL2, even less immunogenicity is seen than withKS-1/4(Construct 3)- IL2. Specifically, during a clinical trial,patients with anti-idiotypic antibodies and antibody directed againstthe antibody-IL2 junction or against the IL-2 moiety are seen at an evenlower frequency than with KS-1/4(Construct 3)-IL2. Antibody variableregions of the invention can also be fused to other cytokines, forexample, interleukins 1, 2, 6, 10, or 12; interferons alpha and beta;TNF, and INF gamma. The invention may be more fully understood byreference to the following non-limiting examples

EXAMPLES Example 1 Methods and Reagents for Expressing KS Antibodies andAssaying their Antigen-Binding Activity

1A. Cell Culture and Transfection

The following general techniques were used in the subsequent Examples.For transient transfection, plasmid DNA was introduced into human kidney293 cells by co-precipitation of plasmid DNA with calcium phosphate[Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.].

In order to obtain stably transfected clones, plasmid DNA was introducedinto the mouse myeloma NS/0 cells by electroporation. NS/0 cells weregrown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum. About 5×10⁶ cells were washed once with PBS andresuspended in 0.5 ml phosphate buffer solution (PBS). Ten μg oflinearized plasmid DNA was then incubated with the cells in a GenePulser. Cuvette (0.4 cm electrode gap, BioRad) for 10 minutes on ice.Electroporation was performed using a Gene Pulser (BioRad) with settingsat 0.25 V and 500 μF. Cells were allowed to recover for 10 minutes onice, after which they were resuspended in growth medium and then platedonto two 96-well plates. Stably transfected clones were selected bygrowth in the presence of 100 nM methotrexate (MTX), which wasintroduced two days post-transfection. The cells were fed every 3 daysfor two to three more times, and MTX-resistant clones appeared in 2 to 3weeks. Supernatants from clones were assayed by anti-human Fc ELISA toidentify high producers [Gillies et al. (1989) J. Immunol. Methods125:191]. High producing clones were isolated and propagated in growthmedium containing 100 nM MTX.

1B. ELISAs

Three different ELISAs were used to determine the concentrations ofprotein products in the supernatants of MTX-resistant clones and othertest samples. The anti-huFc ELISA was used to measure the amount ofhuman Fc-containing proteins, e.g., chimeric antibodies. The anti-hukappa ELISA was used to measure the amount of kappa light chain (ofchimeric or human immunoglobulins). The anti-muFc ELISA was used tomeasure the amount of muFc-containing proteins in test samples (seeExample 1C below).

The anti-huFc ELISA is described in detail below.

A. Coating Plates

ELISA plates were coated with AffiniPure goat anti-human IgG (H+L)(Jackson Immuno Research) at 5 μg/ml in PBS, and 100 μl/well in 96-wellplates (Nunc-Immuno plate Maxisorp). Coated plates were covered andincubated at 4° C. overnight. Plates were then washed 4 times with 0.05%Tween (Tween 20) in PBS, and blocked with 1% BSA/1% goat serum in PBS,200 μl/well. After incubation with the blocking buffer at 37° C. for 2hours, the plates were washed 4 times with 0.05% Tween in PBS and tappeddry on paper towels.

B. Incubation with Test Samples and Secondary Antibody

Test samples were diluted to the proper concentrations in sample buffer,which contained 1% BSA/1% goat serum/0.05% Tween in PBS. A standardcurve was prepared with a chimeric antibody (with a human Fe), theconcentration of which was known. To prepare a standard curve, serialdilutions are made in the sample buffer to give a standard curve rangingfrom 125 ng/ml to 3.9 ng/ml. The diluted samples and standards wereadded to the plate, 100 μl/well, and the plate incubated at 37° C. for 2hours.

After incubation, the plate was washed 8 times with 0.05% Tween in PBS.To each well was then added 100 μl of the secondary antibody, the horseradish peroxidase (MP)-conjugated anti-human IgG (Jackson ImmunoResearch), diluted around 1:120,000 in the sample buffer. The exactdilution of the secondary antibody had to be determined for each lot ofthe HRP-conjugated anti-human IgG. After incubation at 37° C. for 2hours, the plate was washed 8 times with 0.05% Tween in PBS.

C. Development

The substrate solution was added to the plate at 100 μl/well. Thesubstrate solution was prepared by dissolving 30 mg ofo-phenylenediamine dihydrochloride (OPD) (1 tablet) into 15 ml of 0.025M citric acid/0.05M Na₂HPO₄ buffer, pH 5, which contained 0.03% offreshly added H₂O₂. The color was allowed to develop for 30 minutes atroom temperature in the dark. The developing time was subject to change,depending on lot to lot variability of the coated plates, the secondaryantibody, etc. The color development in the standard curve was observedto determine when to stop the reaction. The reaction was stopped byadding 4N H₂SO₄, 100 μl/well. The plate was read by a plate reader,which was set at both 490 nm and 650 nm and programmed to subtract offthe background OD at 650 nm from the OD at 490 nm.

The anti-hu kappa ELISA followed the same procedure as described above,except that the secondary antibody used was horse radishperoxidase-conjugated goat anti-hu kappa (Southern Biotechnology Assoc.Inc., Birmingham, Ala.), used at 1:4000 dilution.

The procedure for the anti-muFc ELISA was also similar, except thatELISA plates were coated with AffiniPure goat anti-murine IgG (H+L)(Jackson Immuno Research) at 5 μg/ml in PBS, and 100 μl/well; and thesecondary antibody was horse radish peroxidase-conjugated goatanti-muIgG, Fcγ (Jackson ImmunoResearch), used at 1:5000 dilution.

1C. Cloning of the KS Antigen (KSA, EpCAM) and Expression of the SolubleForm as Human EpCAM-Murine Fc

Messenger RNA (MRNA) was prepared from LnCAP cells using Dynabeads mRNADirect Kit (Dynal, Inc., Lake Success, NY) according to themanufacturer's instructions. After first strand cDNA synthesis witholigo(dT) and reverse transcriptase, full length cDNA encodingepithelial cell adhesion molecule (also known as KS antigen or KSA), wascloned by polymerase chain reaction (PCR). The sequences of the PCRprimers were based on the published sequence described in Perez andWalker (1989) J. Immunol. 142:3662-3667. The sequence of the senseprimer is TCTAGAGCAGCATGGCGCCCCCGCA (SEQ ID NO: 27), and the sequence ofthe nonsense primer is CTCGAGTTATGCATTGAGTTCCCT (SEQ ID NO: 28), wherethe translation initiation codon and the anti-codon of the translationstop codon are denoted in bold, and the restriction sites XbaI (TCTAGA)and XhoI (CTCGAG) are underlined.

The PCR product was cloned and the correct KSA sequence was confirmed bysequencing several independent clones. The cDNA sequence of the KSA fromLnCAP was essentially identical to the published sequence of KSA fromUCLA-P3 cells (Perez and Walker, 1989). However, at amino acid residuenumber 115, the nucleotide sequence from LnCAP was ATG rather than ACG(Met instead of Thr), and at amino acid residue number 277, thenucleotide sequence from LnCAP was ATA rather than ATG (Ile instead ofMet).

Binding of KS-1/4 antibody to recombinant KSA was demonstrated byimmunostaining. Surface expression of KSA was obtained by transfectingcells, e.g., CT26, B16, etc., with full length KSA in a suitablemammalian expression vector (pdCs, as described in U.S. Pat. No.5,541,087), followed by immunostaining with the KS-1/4 antibody. For theexpression of KSA as a soluble antigen, the portion of the cDNA encodingthe transmembrane domain of the KSA was deleted. To facilitate.expression, detection, and purification, the soluble KSA was expressedas a KSA-muFc, the construction of which is described as follows. The780 by XbaI-EcoRI restriction fragment encoding the soluble KSA wasligated to the AfIII-XhoI fragment encoding the muFc (U.S. Pat. No.5,726,044) via a linker-adaptor:

5′ AA TTC TCA ATG CAG GGC 3′ (SEQ ID NO: 29) 3′ G AGT TAC GTC CCG AAT T5′ (SEQ ID NO: 30)

The XbaI-XhoI fragment encoding soluble KSA-muFc was ligated to the pdCsvector. The resultant expression vector, pdCs-KSA-muFc, was used totransfect cells and stable clones expressing KSA-muFc were identified byanti-muFc ELISA.

1D. Measurement of Antigen Binding

KSA-muFc in conditioned medium was first purified by Protein Achromatography according to supplier's protocol (Repligen, Cambridge,Mass.). Purified KSA-muFc was used to coat 96-well plates (Nunc-Immunoplate, Maxisorp) at 5 μg/ml in PBS, and 100 μl/well. The assay wassimilar to the ELISA procedure described in Example 1B. Briefly, coatedplates were covered and incubated at 4° C. overnight. Plates then werewashed and blocked. Test samples were diluted to the properconcentrations in the sample buffer, added to the plate at 100 μl/well,and the plate was incubated at 37° C. for 1 hour. After incubation, theplate was washed 8 times with 0.05% Tween in PBS. To each well was thenadded 100 μl of the secondary antibody, the horse radishperoxidase-conjugated anti-human IgG (Jackson Immuno Research), dilutedaround 1:120,000 in the sample buffer. The plate was then developed andread as described in Example 1B.

1E. Measurement of on-Rates and Off-Rates of KS-1/4 Antibodies fromEpCAM using a Biacore Assay.

The affinity of KS-1/4 and KS-IL2 molecules for the antigen EpCAM weremeasured by surface plasmon resonance analysis of the antibody-antigeninteraction, using a Biacore machine (Biacore International AB, Uppsala,Sweden). EpCAM-murineFc was coupled to a CM5 sensor chip using an aminecoupling protocol supplied by the manufacturer. KS-1/4 and KS-IL2 atconcentrations varying between 25 nm and 200 nM were then passed overthe chip, whereby binding to the chip was observed. Using the built-incurve-fitting routines of the Biacore software, the on-rate, off-rate,association and dissociation constants were calculated.

1F. Measurement of Binding Affinities of KS-1/4 Antibodies Using CellLines Expressing EpCAM

Purified KS-1/4 antibodies were iodinated with ¹²⁵I using standardtechniques, and increasing concentrations of labeled protein wereincubated with the EpCAM-positive cell line PC-3. Saturation bindingcurves were generated and the dissociation constants were determined byScatchard analysis.

Example 2 Cloning of cDNAs Encoding V_(H) and V_(K) of mouse KS-1/4 andConstruction of Vector for the Expression of KS-1/4 Hybridoma-DerivedAntibody

Messenger RNA prepared from the mouse KS-1/4-expressing hybridoma(obtained from R. Reisfeld, Scripps Research Institute) was reversetranscribed with oligo(dT) and then used as templates for PCR to amplifythe sequences encoding the variable region of the heavy chain (V_(H))and the variable region of the light chain (V_(K)). The PCR primers weredesigned based on published sequences (Beavers et al., ibid.). The PCRprimers for V_(H) had the following sequences:

(SEQ ID NO: 31) V_(H) forward primer (5′) GACTCGAGCCCAAGTCTTAGACATC (3′)(SEQ ID NO: 32) V_(H) reverse primer (5′)CAAGCTTACCTGAGGAGACGGTGACTGACGTTC (3′),where the CTCGAG and AAGCTT sequences represent the XhoI and HindHIIIrestriction sites, respectively, used for ligating the V_(H) into theexpression vector (see below); and the TAC in the reverse primer wouldintroduce GTA, the splice donor consensus sequence, in the sense strandof the PCR product.

The PCR primers for V_(K) had the following sequences:

(SEQ ID NO: 33) V_(K) forward primer (5′) GATCTAGACAAGATGGATTTTCAAGTG(3′) (SEQ ID NO: 34) V_(K) reverse primer (5′)GAAGATCTTACGTTTTATTTCCAGCTTGG (3′)where the TCTAGA and AGATCT sequences represent the XbaI and BgIIIrestriction sites, respectively, used for ligating the V_(K) into theexpression vector (see below); ATG is the translation initiation codonof the light chain; and the TAC in the reverse primer would introduceGTA, the splice donor consensus sequence, in the sense strand of the PCRproduct.

The PCR products encoding the V_(H) and V_(K) of the mouse KS-1/4antibody were cloned into pCRII vector (Invitrogen, Carlsbad, Calif.).Several V_(H) and V_(K) clones were sequenced and the consensus sequenceof each determined. The V_(H) and V_(K) sequences were inserted in astepwise fashion into the expression vector pdHL7. The ligations tookadvantage of the unique XhoI and HindIII sites for the V_(H), and theunique XbaI and Bg1II/BamHI sites for the V_(K) (the unique Bg1II in theV_(K) insert and the unique BamHI in the vector have compatibleoverhangs). The resultant construct is called pdHL7-hybridoma chKS-1/4,which already contained transcription regulatory elements and human Igconstant region sequences for the expression of chimeric antibodies(Gillies et al. (1989) J. Immunol. Methods 125:191).

The expression vector pdHL7 was derived from pdHL2 [Gillies et al.(1991) Hybridoma 10:347-356], with the following modifications: in theexpression vector pdHL2, the transcriptional units for the light chainand the heavy chain-cytokine consisted of the enhancer of the heavychain immunoglobulin gene and the metallothionein promoter. In pdHL7,these two transcriptional units consisted of the CMV enhancer-promoter[Boshart et al. (1985) Cell 41:521-530]. The DNA encoding the CMVenhancer-promoter was derived from the Afl111-HindIII fragment of thecommercially available pcDNAI (Invitrogen Corp., San Diego, Calif.).

Example 3 Expression Studies of Murine KS-1/4 Antibodies

This example discusses expression studies performed using an antibodyexpression plasmid encoding the V region sequences disclosed in U.S.Pat. No. 4,975,369.

3A. Plasmid Construction

To directly compare the chimeric antibodies encoded by the HybridomaKS-1/4 sequence and those sequences described in U.S. Pat. No.4,975,369, the cDNA encoding the VH sequence described in U.S. Pat. No.4,975,369 was synthesized. This was then ligated into the pdHL7expression vector already containing the V_(K) of KS-1/4.

In order to construct the V_(H) sequence described in U.S. Pat. No.4,975,369, an NdeI-HindIII fragment encoding part of the V_(H) sequencewas obtained by total chemical synthesis. Overlapping oligonucleotideswere chemically synthesized and ligated. The ligated duplex was thensubcloned into a XbaI-HindIII pBluescript vector (Stratagene, LaJolla,Calif.).

This DNA encodes the protein sequence IQQPQNMRTM of U.S. Pat. No.4,975,369. Immediately 3′ to the coding sequence is the splice donorsite beginning with gta. The ctag at the 5′ end of the top strand is theoverhang for the XbaI cloning site. The XbaI site was created only forcloning into the polylinker of the pBluescript vector. It was followedimmediately by the NdeI restriction site (CATATG). The agct at the 5′end of the bottom strand is the overhang of the HindIII cloning site.This HindIII sticky end is later ligated to the HindIII site in theintron preceding the C-γl gene [Gillies et al. (1991) Hybridoma10:347-356].

After sequence verification, the NdeI-HindIII restriction fragment wasisolated. This, together with the XhoI-NdeI fragment encoding theN-terminal half of V_(H), was then ligated to the XhoI-HindIII digestedpdHL7 expression vector containing the V_(K) of KS-1/4. The resultantconstruct, pdHL7-'369 chKS-1/4, contained the V_(K) and V_(H) describedin U.S. Pat. No. 4,975,369 (referred to as U.S. Pat. No. 4,975,369chKS-1/4).

3B. Comparison of Hybridoma chKS-1/4 and U.S. Pat. No. 4,975,369chKS-1/4 Antibodies

The plasmid DNAs pdHL7-hybridoma chKS-1/4 and pdHL7-'369 chKS-1/4 wereintroduced in parallel into human kidney 293 cells by the calciumphosphate coprecipitation procedure mentioned above. Five dayspost-transfection, the conditioned media were assayed by anti-huFc ELISAand kappa ELISA (see Example 1 for ELISA procedures) and the results aresummarized in Table 1.

TABLE 1 Antibody huFc ELISA Kappa ELISA Hybridoma chKS-1/4 254 ng/mL 200ng/mL US4,975,369 chKS-1/4  14 ng/mL  0 ng/mL

The results indicated that hybridoma chKS-1/4 was expressed and secretednormally, and that the secreted antibody consisted of roughly equimolaramounts of heavy and light chains, within the accuracies of the twodifferent ELISAs. On the other hand, only a low level of heavy chain wasdetected in the conditioned medium for the U.S. Pat. No. 4,975,369chKS-1/4 antibody, and no kappa light chain was associated with it.

Western blot analysis was performed on the total cell lysates and theconditioned media of the two transiently transfected cell lines. Theprocedures for Western blot analysis were as described in (Sambrook etal. (1989), supra). In order to analyze the total cell lysates, thetransfected cells were lysed, centrifuged to remove the debris; and thelysate from the equivalent of 5×10⁵ cells applied per lane. To analyzethe conditioned media, the protein product from 300 μL of theconditioned medium was first purified by Protein A Sepharosechromatography prior to SDS-PAGE under reducing conditions. AfterWestern blot transfer, the blot was hybridized with a horseradishperoxidase-conjugated goat anti-human IgG, Fcγ(Jackson ImmunoResearch),used at 1:2000 dilution.

The Western blot transfer showed that under the conditions used, theheavy chain was detected in both the conditioned media and the lysedcells of the transfection with pdHL7-hybridoma chKS-1/4. This resultindicates that the heavy chain of the chKS-1/4 antibody was produced inthe cells and secreted efficiently (together with the light chain). Onthe other hand, the heavy chain from the transfection with pdHL7-'369chKS-1/4 was detected only in the cell lysate but not in the conditionedmedia. This result indicated that although a comparable level of heavychain was produced inside the cell, it was not secreted. This findingwas Consistent with the ELISA data, which showed that there was no kappalight chain associated with the small amount of secreted heavy chain inthe U.S. Pat. No. 4,975,369 chKS-1/4 antibody. It is understood thatimmunoglobulin heavy chains typically are not normally secreted in theabsence of immunoglobulin light chains [Hendershot et al. (1987)Immunology Today 8:111].

In addition to the foregoing, NS/0 cells were transfected byelectroporation with the plasmids pdHL7-Hybridoma chKS-1/4 andpdHL7-U.S. Pat. No. 4,975,369 chKS-1/4 in parallel. Stable clones wereselected in the presence of 100 nM MTX, as described in Example 1, andthe conditioned media of the MTX-resistant clones in 96-well plates wasassayed by anti-huFc ELISA, as described in Example 1. The results aresummarized in Table 2.

TABLE 2 Total number of Highest level Antibody clones screened Mode* ofexpression* Hybridoma chKS-1/4 80 0.1-0.5 μg/mL (41) 10-50 μg/mL (4)US4,975,369 chKS-1/4 47 0-10 ng/mL (36) 0.1-0.4 μg/mL (4) (*The numbersin parentheses denote the number of clones in the mode or the numberexpressing the highest levels of product, as determined by anti-FcELISA.)

When screened at the 96-well stage, the majority of the clones obtainedwith the pdHL7-hybridoma chKS-1/4 construct produced about 100 ng/mL to500 ng/mL of antibody, with the best clones producing about 10-50 μg/mL.On the other hand, the majority of the clones obtained with thepdHL7-'369 chKS-1/4 construct produced about 0 ng/mL to 10 ng/mL ofantibody, with the best producing about 300-400 ng/mL. To examine thecomposition and binding properties of the U.S. Pat. No. 4,975,369chKS-1/4 antibody, it was necessary to grow up the clones that producedat 300-400 ng/mL. Two of these clones were chosen for expansion.However, their expression levels were found to be very unstable. By thetime the cultures were grown up to 200 mL, the expression levels of bothclones had dropped to about 20 ng/mL, as assayed by anti-Fc ELISA. Whenthe same conditioned media were assayed by the anti-kappa ELISA, nokappa light chain was detected, as was the case in transient expressionin 293 cells.

The following experiment indicated that no detectable kappa light chainwas associated with the U.S. Pat. No. 4,975,369 chKS-1/4 heavy chain.Briefly, 50 mL each of the conditioned media from each of the clones wasconcentrated by Protein A chromatography. The eluate were assayed byanti-Fc ELISA and anti-kappa ELISA. As a control, conditioned mediumfrom a hybridoma chKS-1/4-producing clone was treated the same way andassayed at the same time. The ELISA results are summarized in Table 3.

TABLE 3 Antibody huFc ELISA Kappa ELISA Hybridoma chKS-1/4 42 μg/mL 44μg/mL US4,975,369 chKS-1/4-clone 1 253 ng/mL 0 ng/mL US4,975,369chKS-1/4-clone 2 313 ng/mL 0 ng/mL

The results showed that there was indeed no detectable kappa light chainassociated with the U.S. Pat. No. 4,975,369 chKS-1/4 heavy chain.Furthermore, the hybridoma chKS-1/4 antibody was shown to bind KSantigen at 10-20 ng/mL, whereas the U.S. Pat. No. 4,975,369 antibodyfrom both clones and concentrated to 253 and 313 ng/mL, still did notbind KS antigen (see Example 9 for measurement of binding to KSantigen.)

Example 4 Expression and Characterization of Variant KS Antibodies

Mutations that significantly lower the expression or the affinity of anantibody for a target molecule are expected to be less effective fortherapeutic purposes in humans. Some approaches to reducingimmunogenicity, such as “veneering,” “humanization,” and“deimmunization” involve the introduction of many amino acidsubstitutions, and may disrupt binding of an antibody to an antigen(see, e.g., U.S. Pat. Nos. 5,639,641; and 5,585,089; and PCT PublicationNos. WO 98/52976; WO 00/34317). There is a need in the art for classesof antibody sequences that will bind to epithelial cell adhesionmolecule, but which are distinct from the original mouse monoclonalantibodies that recognize this antigen.

Various combinations of KS-1/4 heavy and light chain variable (“V”)regions were tested for their ability to be expressed, and for theirability to bind to EpCAM. These results are summarized in Tables 4-6 anddescribed below.

TABLE 4 Sequences of KS-1/4 antibody heavy and light chain V regions.Light chains:        10        20        30        40        50        60         |         |         |         |         |         | VK0QILLTQSPAIMSASPGEKVTMTCSASSSVSYMLWYQQKPGSSPKPWIFDTSNLASGFPAR VK1 QI VLTQSPA SLAV SPG QRA T I TCSASSSVSY I LWYQQKPG Q PPKPWIFDTSNLASGFP S RVK6 E I V LTQSPA TL S L SPGE R VT L TCSASSSVSYMLWYQQKPG Q APK LLIFDTSNLASG I PAR VK7 QILLTQSPAIMSASPGE RVTMTCSASSSVSYMLWYQQKPGSSPKPWIFDTSNLASGFPAR VK8 E I V LTQSPA TL S L SPGER VT L TCSASSSVSYMLWYQQKPGSSPKPWIFDTSNLASGFPAR        70        80        90        100         |         |         |         | VK0FSGSGSGTSYSLIISSMEAEDAATYYCHQRSGYPYTFGGGTKLEIK (SEQ ID NO: 1) VK1FSGSGSGTSYTL T I N S L EAEDAATYYCHQRSGYPYTFGGGTK V EIK (SEQ ID NO: 11)VK6 FSGSGSGT D YTL T ISS L E P ED F A V YYCHQRSGYPYTFGGGTKLEIK (SEQ IDNO: 7) VK7 FSGSGSGTSYSLIISSME P EDAATYYCHQRSGYPYTFGGGTKLEIK (SEQ ID NO:8) VK8 FSHSGSGTSYSLIISSMEAEDAATYYCHQRSGYPYTFGGGTKLEIK (SEQ ID NO: 9)Heavy chains:        10        20        30        40        50        60         |         |         |         |         |         | VH0QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQTPGKGLKWMGWINTYTGEPTY VH1QIQLVQSGPELKKPG SS VKISCKASGYTFTNYGMNWV R Q A PGKGLKWMGWINTYTGEPTY VH2QIQLVQSGPELKKPG SS VKISCKASGYTFTNYGMNWV R Q A PGKGLKWMGWINTYTGEPTY VH2.5QIQLVQSGPELKKPG SS VKISCKASGYTFTNYGMNWV R Q A PGKGLKWMGWINTYTGEPTY VH6 QV QLVQSG A EVKKPGE S VKISCKASGYTFTNYGMNWV R Q A PGKGL E WMGWINTYTGEPTYVH7 QIQLVQSG A EVKKPGETVKISCKASGYTFTNYGMNWVKQTPGKGLKWMGWINTYTGEPTY VH369QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQTPGKGLKWMGWINTYTGEPTY        70        80         90        100        110         |         |          |         |         | VH0ADDFKGRFAFSLETSASTAFLQINNLRNE.DMATYFCVRFISKGDYWGQGTSVTVSS (SEQ ID NO: 2)VH1 ADDFKGRF TITA ETSTST LY LQ L NNLR S E.D T ATYFCVRF M SKGDYWGQGT TVTVSS (SEQ ID NO: 21) VH2 ADDFKGRF TITA ETSTST LY LQ L NNLR S E.D TATYFCVRF I SKGDYWGQGT T VTVSS (SEQ ID NO: 22) VH2.5 ADDFKGRF TITA ETSTSTLY LQ L NNLR S E.D T ATYFCVRF I SKGDYWG T GT T VTVSS (SEQ ID NO: 19) VH6A QK F Q GR VTI SL D TSTSTA Y LQ LSS LR A E.D T A VYFCVRFISKGDYWGQGTSVTVSS (SEQ ID NO: 17) VH7 ADDFKGRFAFSLETSTSTAFLQINNLRS E.D T ATYFCVRFISKGDYWGQGTSVTVSS (SEQ ID NO: 18) VH369ADDFKGRFAFSLETSASTAFLQI qqpqnmrt MATYFCVRFISKGDYWGQGTSVTVSS (SEQ ID NO:35)

TABLE 5 Sequences of KS 1/4 antibody variants and CDR3 heavy chainvariants with single amino acid insertions. VH2 partial seq.: . . .ATYFCVRF I S K GDYWGQG . . . (amino acid residues 92-109 of SEQ ID NO:22) VH2.1: . . . ATYFCVRF IIS K GDYWGQG . . . (SEQ ID NO: 36) VH2.2: . .. ATYFCVRF IVS K GDYWGQG . . . (SEQ ID NO: 37) VH2.3: . . . ATYFCVRF ISAK GDYWGQG . . . (SEQ ID NO: 38) VH2.4: . . . ATYFCVRF I S KTGDYWGQG .. . (SEQ ID NO: 39)

TABLE 6 Expression levels and binding activity of variant KS-1/4antibodies. Expression EpCAM affinity Transient (*) Stable (*) RelativeConstruct (in ng/mL) (in μg/mL) binding (**) Kd (nM) Group 1 VK0/VH0(Hybridoma chKS-1/4) 10-50 1x 1.0 × 10⁻⁹ VK0/VH′369 (′369 chKS-1/4)   0.1-0.4 (***) >>30x   VK8/VH7 (Construct 3) 10-50 1.0 × 10⁻⁹ VK6/VH6(Construct 1) 300 n.d. VK7/VH7 (Construct 2) 30 VK8/VH7-IL2 10-50 1.0 ×10⁻⁹ VK1/VH1-IL2 10-50 7.9 × 10⁻⁹ VK1/VH2-IL2 10-50 3.1 × 10⁻⁹ Group 2VK8/VH7 (Construct 3; control) 1500 1x VK0/VH1 1500 8x VK1/VH7 1500 1xVK1/VH1 1500 2x VK1/VH2 1500 1x-2x VK1/VH1-IL2 1500 5x VK1/VH2-IL2 15001.5x  VK1/VH2.5-IL2 1500 3x-4x Group 3 VK8/VH7-IL2 (control) 760 1xVK1/VH1-IL2 350 2x VK1/VH2.1-IL2 290 >10x  VK1/VH2.2-IL2 270 >10x VK1/VH2.3-IL2 190 7x VK1/VH2.4-IL2 210 3x (*) Routinely achievablelevels. (**) “Relative Binding” is expressed as the fold-increase inprotein concentration required to reach an equivalent level of binding.Thus, a larger number reflects a lower affinity for EpCAM. (***) Kappalight chain was not detectable by ELISA (equivalent to background);therefore, functional antibodies were not expressed. (****) n.d. = notdetectable In Group 2 and Group 3, the relative binding activity of eachprotein was normalized to the control shown in the first line for thatgroup. The ELISA assay is primarily a reflection of off-rates, based onamount of protein bound after several rounds of washes. It is used as arapid screen to rule out poor binders, but is not a precise measure ofaffinity. In Group 3, VH2 variants VH2.1-VH2.4 were compared with VH1 todetermine if amino acid insertions might result in improved relativebinding.

The sequences are related as follows. As described in the examples, theVH0 and VK0 sequences were derived from PCR amplification from ahybridoma cell line that expresses the original mouse-derived KS-1/4(SEQ ID NO: 1 and SEQ ID NO: 2). VH-369 is the VH sequence disclosed inU.S. Pat. No. 4,975,369. Sequences VH1, VH2, VH2.1-2.4 VK1, and VK2 werederived either using deimmunization technology where potential T cellepitopes are eliminated or weakened by introduction of mutations thatreduce binding of a peptide epitope to an MHC Class II molecule, or bychanging non-human T cell epitopes so that they correspond to humanself-epitopes that are present in human antibodies. The design of theseconstructs is further described and analyzed below. Constructs of Table6 were generated by transfecting mammalian cells with combinations ofnucleic acids that expressed the corresponding heavy and light chain V.regions. Sequences VH6, VH7, VK6, VK7, and VK8 were generated bychanging surface residues of the hybridoma KS-1/4 to human counterpartsas described below, with the purpose of removing potential human B cellepitopes. Constructs 1 through 3 were generated by transfectingmammalian cells with combinations of nucleic acids that expressed heavyand light chain V regions VH6, VH7, VK6, VK7, and VK8 as described inTable 4 and below.

4A. Characterization of KS Antibodies with Fewer Human T Cell Epitopes

Sequences VH2.1-VH2.5 were made to test whether certain amino acidinsertions and substitutions in the region of the KS-1/4 heavy chainCDR3 could be tolerated. Expression vectors for the light and heavychain combinations VK0/VH1, VK1/VH7, VK1/VH1, VK1/VH2, VK1/VH1-IL2,VK1/VH2-IL2, and VK1/VH2.5-IL2 were constructed and the correspondingantibodies and antibody-IL2 fusion proteins expressed and testedaccording to methods described in the preceding examples.

Specifically, sequences VH1, VH2, VK1, and VK2 were obtained by totalchemical synthesis. For each of these sequences, a series of overlappingoligonucleotides that span the entire coding and complementary strandsof these regions were chemically synthesized, phosphorylated, andligated. The ligated duplex molecules were then amplified by PCR withappropriate primers to the fragment ends, introduced into pCRII vector(Invitrogen, Carlsbad, Calif.) and the sequences verified. These DNAfragments were then introduced into the expression vector pdHL7 atappropriate sites to generate the complete heavy (“H”) chain and light(“L”) chain, respectively.

Sequence VH2.5 was derived from VH2 by the modification of a singlecodon to obtain a Thr rather than a Gln at position 108 (Table 4), usingstandard molecular biology techniques.

The antibodies were tested by ELISA (Table 6) and using surface plasmonresonance (Biacore machine and software) to compare their ability tobind to EpCAM. Results of the ELISA experiments were considered toreflect primarily off-rate and not on-rate, and to be generally lessprecise, such that a poor ELISA result was generally used to excludecertain constructs from further consideration. However, antibodies thatshowed good binding by the ELISA test needed to be characterizedfurther.

Results of the surface plasmon resonance analysis were as follows:

Fusion Protein k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹) K_(D) (M) VK8/VH7-IL2 3.1× 10⁵ 3.2 × 10⁻⁴ 1.0 × 10⁻⁹ VK1/VH2-IL2 1.7 × 10⁵ 5.3 × 10⁻⁴ 3.1 × 10⁻⁹VK1/VH1-IL2 2.8 × 10⁵ 2.2 × 10⁻³ 7.9 × 10⁻⁹

Because the off-rate of VK1/VH1-IL2 was much faster than for VK1/VH2-IL2or VK8/VH7-IL2, VK1/VH1-IL2 was considered to be a less useful fusionprotein.

Considering that VK1/VH1-IL2 and VK1/VH1-IL2 differ only by themethionine/isoleucine difference at V_(H) position 100 in CDR3, theenhanced off-rate of VK1/VH1-IL2 compared to VK1/VH2-IL2 suggests thatthis position makes a hydrophobic contact with EpCAM, and that theslightly longer methionine side-chain makes a less effective contact. Inthe field of protein-protein interactions, it is generally thought thathydrophobic interactions play a major role in determining off-rates buta much less significant role in determining on-rates.

4B. Characterization of KS-1/4 Variants with Single Amino AcidInsertions

The importance of the CDR3 sequence in the heavy chain V region for theaffinity of the KS antibody to EpCAM was determined with a series ofvariants that contained an amino acid insertion or substitution in thisregion. Sequences VH2.1, VH2.2, VH2.3, and VH2.4 were generated bymanipulation of an expression vector encoding VH2 and VK. using standardrecombinant DNA techniques. The resulting expression vectors weretransfected into NS/0 cells and secreted antibody proteins purified asdescribed in preceding examples.

It was found that the VH1 variant was suboptimal compared to the VH2variant, indicating that the isoleucine in CDR3 could not be substitutedwith methionine. The next goal was to test whether insertion of an aminoacid in CDR3 could yield a KS-1/4 heavy chain V region with betterbinding characteristics than VH1. The data in Table 6 compare thebinding of VK1/VH2.1, VK1/VH2.2, VK11VH2.3, and VK1/VH2.4, with VK1/VH1.It was found that none of the constructs with an amino acid insertion inthe KS-1/4 V_(H) CDR3 showed improved antigen binding compared to VH1,rather, antigen binding activity of the insertion mutants was eithersomewhat decreased or profoundly decreased.

These results indicate that insertion of amino acids in CDR3 generallyis deleterious to the antigen binding activity of KS-1/4 heavy chain Vregions. When this data is analyzed, some general conclusions emerge.Specifically, the segment of KS-1/4 V_(H) amino acid at positions 84 to108, consisting of the amino acidsAsn-Asn-Leu-Arg-Asn-Glu-Asp-Met-Ala-Thr-Tyr-Phe-Cys-Val-Arg-Phe-Ile-Ser-Lys-Gly-Asp-Tyr-Trp-Gly-Gln,is important for KS-1/4 antigen binding. This segment includes aframework segment,Asn-Asn-Leu-Arg-Asn-Glu-Asp-Met-Ala-Thr-Tyr-Phe-Cys-Val-Arg, which isgenerally tolerant to single and multiple amino acid substitutions, butnot tolerant to amino acid insertions, which may have a deleteriouseffect on expression and assembly. In addition, the data suggests thatfor the amino acids at positions 86, 91, 93, 94, and 95, it ispreferable to have hydrophobic amino acids for an antibody that isefficiently expressed and binds to EpCAM.

Insertion of an amino acid in the V_(H) CDR3 segment, consisting ofPhe-Ile-Ser-Lys-Gly-Asp-Tyr, is generally deleterious to the EpCAMantigen-binding function of a KS-1/4 antibody, although some insertionscan be tolerated with only partial loss of activity. Similarly,substitution of these positions is also generally deleterious to bindingof the EpCAM antigen, although some insertions can be tolerated withonly partial loss of activity.

4C. Construction of Active Derivatives of KS-1/4 Antibodies with MouseSurface Residues Converted to their Human Counterparts

Antibodies were prepared by substituting amino acids within the KS-1/4antibody with amino acids commonly found in human antibodies in order tominimize the immunogenicity of the mouse-derived V regions. Preferred KSderivatives also retained specific binding affinity for human EpCAM.

Construct 1. It was found that the KS-1/4 light chain most closelyresembled human consensus subgroup III, and the heavy chain most closelyresembled subgroup I. Based on these similarities, a conceptual sequenceconsisting of the human consensus subgroup amino acids andKS-1/4-derived CDRs and non-consensus amino acids was generated. Forthis and the following constructs a three-dimensional model wasgenerated using a Silicon Graphics Workstation and BioSym molecularmodeling software.

Inspection of the three-dimensional model revealed that certainhuman-derived amino acids were close to the CDRs and were likely toinfluence their conformation. Based on this analysis, in the lightchain, human Ser22, Arg44, and Phe66 were changed back to Thr, Lys, andTyr, respectively. In the heavy chain, it was believed such changes wereunnecessary. In the final design for Construct 1, the light chain had 18human amino acids not found in the mouse light chain, and the heavychain had 22 human amino acids not found in the mouse heavy chain.

DNAs for expression of Construct 1 were created using syntheticoligonucleotides. The Construct 1 protein was efficiently expressed butwas found to be more than 10-fold less active in an EpCAM binding assay.

Construct 2. A less aggressive approach was then taken, by which onlythe following changes were introduced:

Light chain: K18R, A79P

Heavy chain: P9A, L11V, A76T, N88S, M91T

DNAs for expression of Construct 2 were created using syntheticoligonucleotides and standard recombinant DNA techniques. The Construct2 protein was not efficiently expressed. It was further found that thecombination of Construct 2 light chain and mouse KS-1/4 heavy chain wasnot efficiently expressed, while the combination of Construct 2 heavychain and mouse KS-1/4 light chain was efficiently expressed. Thus, theexpression defect appeared to lie in the Construct 2 light chain.

Construct 3. Based on the apparent expression defect in the Construct 2light chain, a new light chain was constructed by fusing the N-terminalportion of the light chain of Construct 1 with the C-terminal portion ofthe mouse light chain. The KpnI site, which encodes the amino acids atpositions 35 and 36, was used. When this light chain was combined withthe Construct 2 heavy chain, efficient expression and no significantloss of binding was observed.

Because Construct 3 resulted in an antibody with superior properties interms of protein expression and affinity for the antigen when comparedto Construct 1 or 2, DNA sequences of Construct 3 were inserted intopdHL7s−μL2, resulting in pdHL7s-VK8/VH7-IL2, which is disclosed as SEQID NO: 40. For expression purposes, this plasmid DNA was electroporatedinto mouse myeloma cells NS/0 to produce a stably transfected cell lineas described in Example 1A. Culture medium taken from stable clones wasthen assayed for antibody expression in an ELISA coated with human Fc,as described in Example 1B. The amino acid sequences of the heavy andlight chain for this antibody fusion protein are shown in SEQ ID NO: 41and SEQ ID NO: 42, respectively.

In addition, the binding of iodinated VK8/VH7 and VK8/VH7-IL2 to EpCAMexpressed on the surface of PC-3 tumor cells was compared to binding ofiodinated VK0/VH0-IL2, using methods described in Example 1F. Withinexperimental error, essentially identical binding affinities were foundfor VK8/VH7 and VK0/VH0, and for VK8/VH7-IL2 and VK0/VH0-IL2.

4D. Structure-Function Relationships Useful in Constructing ActiveKS-1/4 Antibodies

Taken together, the antigen binding activities of KS-1/4 antibodies andfusion proteins with the disclosed V region sequences provide guidancein designing sequences of KS-1/4 antibodies to EpCAM, as well as forproper expression and secretion of KS-1/4 antibodies. In particular, theKS-1/4 heavy and light chain V regions can tolerate multiple amino acidsubstitutions and retain activity, provided that these amino acidsubstitutions are outside the CDRs. The KS-1/4 heavy and light chain Vregions do not generally appear to tolerate amino acid insertions,especially within CDRs or in framework regions between CDRs.

For example, if the hybridoma KS-1/4 sequence is taken to be a starting,“wild-type” sequence, the data indicate that the heavy chain V regioncan tolerate amino acid substitutions at positions 9, 11, 16, 17, 38,40, 69, 70, 71, 72, 76, 79, 80, 83, 88, 91, and 111 with little or noloss of activity. Similarly, the light chain can tolerate amino acidsubstitutions at positions 1, 3, 10, 11, 12, 13, 17, 18, 19, 21, 41, 42,59, 71, 73, 75, 77, and 103 with little or no loss of activity. Thesechanges are outside the CDRs of ICS-1/4 heavy and light chain V regions.The 17 clearly acceptable heavy chain amino acid substitutions representabout 21% of the amino acid positions outside the CDRs, and about 68% ofthe amino acid positions outside the CDRs for which an amino acidsubstitution was attempted. Similarly, the eighteen clearly acceptablelight chain amino acid substitutions represent about 23% of the aminoacid positions outside the CDRs, and about 72% of the amino acidpositions outside the CDRs for which an amino acid substitution wasattempted. There were only two examples of an amino acid substitutionoutside of a CDR that resulted in a significantly less useful protein:the substitution Ala79Pro in the light chain, which appeared to have anegative impact on expression; and the substitution Q108T in the heavychain, which had a negative impact on antigen binding. Thus, an aminoacid substitution can be introduced into a KS-1/4 antibody heavy chainor light chain sequence outside of a CDR, and there is a highprobability that the substitution will result in an active protein.

Mutations involving the substitution of an amino acid in a CDR oftenhave a negative impact on antigen binding. For example, the substitutionI100M in the heavy chain reduces binding by about 8-fold. Mutations thatinvolve the insertion of an amino acid generally have a negative impacton the utility of a KS-1/4 sequence. For example, the VH-'369 heavychain V region is unable to assemble into a proper antibody with a lightchain, as described herein. The VH2.1 to 2.4 mutations have an insertionof an amino acid in CDR3 of the heavy chain V region, and each of thesemutations has a negative impact on antigen binding.

Example 5 Immunogenicity of a KS Antibody (Construct 3)-IL2 FusionProtein in Humans

In a human clinical trial, twenty two patients received one or moretreatment regimes, with each treatment regime comprising threeconsecutive daily 4-hour intraveous infusions of KS antibody (Construct3)-IL2. Each treatment regime was separated by a month (Weber et al.(2001). Proc. Am. Soc. Clin. Oncology 20:259a.). Serum samples wereharvested from each patient before and after each treatment regime andtested for antibody reactivity against the whole KS Antibody (Construct3)-IL2 molecule or the Fc-IL2 component (without the Fv region). Noreactivity was observed in any of the pre-immune sera. The resultsindicated that only 4 patients experienced any significant immuneresponse against either the Fv regions alone, or both the Fv regions andthe Fc-IL2 component. Furthermore, these responses did not appear to beboosted upon subsequent exposure to huKS-IL2.

It is believed that the use of the antibody-IL2 fusion proteinconstitutes a particularly stringent test of the immunogenicity of the Vregion, because the interleukin-2 moiety has an adjuvant effect.Accordingly, the results indicate that the KS Antibody (Construct 3) maybe administered to humans with only a small number of recipientsapparently developing an antibody response to the KS antibody (Construct3)-IL2 fusion protein. These results are particularly encouraging inview of the fact that the KS antibody (Construct 3) contains a variableregion that is almost entirely murine in origin but with a few aminoacid residues replaced with the corresponding human amino acid residues.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

The disclosure of each of the patent documents and scientificpublications disclosed herein, are incorporated by reference into thisapplication in their entirety.

1-18. (canceled)
 19. A nucleic acid encoding an anti-EpCAM proteincomprising an antibody that binds human EpCAM and that comprises: (a) anamino acid sequence selected from the group consisting of: (i) aminoacid residues 24-31 of SEQ ID NO: 1; (ii) amino acid residues 49-55 ofSEQ ID NO: 1; and (iii) amino acid residues 88-96 of SEQ ID NO: 1; theantibody further comprising (b) an amino acid sequence defining animmunoglobulin light chain framework region selected from the groupconsisting of: (i) amino acid residues 1-23 of SEQ ID NO:8; and (ii)amino acid residues 1-23 of SEQ ID NO:9.
 20. The nucleic acid of claim19, wherein the antibody comprises amino acids 1-106 of SEQ ID NO:9. 21.The nucleic acid of claim 19, wherein the antibody has a Kd for EpCAM ofat least 10⁻⁸ M.
 22. The nucleic acid of claim 19, wherein the proteincomprises a cytokine.
 23. The nucleic acid of claim 22, wherein thecytokine is IL-2.
 24. A cell comprising the nucleic acid of claim 19.25. A nucleic acid encoding an anti-EpCAM protein, wherein the proteincomprises an antibody that binds human EpCAM and that comprises: (a) anamino acid sequence selected from the group consisting of: (i) aminoacid residues 26-35 of SEQ ID NO:2; (ii) amino acid residues 50-62 ofSEQ ID NO:2; and (iii) amino acid residues 101-105 of SEQ ID NO:2; theantibody further comprising (b) an amino acid sequence defining animmunoglobulin heavy chain framework region selected from the groupconsisting of: (i) amino acid residues 1-25 of SEQ ID NO: 18; and (ii)amino acid residues 67-98 of SEQ ID NO:
 18. 26. The nucleic acid ofclaim 25, wherein the antibody comprises amino acids 1-116 of SEQ IDNO:18.
 27. The nucleic acid of claim 25, wherein the antibody has a Kdfor EpCAM of at least 10⁻⁸ M.
 28. The nucleic acid of claim 25, whereinthe protein comprises a cytokine.
 29. The nucleic acid of claim 28,wherein the cytokine is IL-2.
 30. A cell comprising the nucleic acid ofclaim
 25. 31. A nucleic acid encoding an anti-EpCAM protein, wherein theprotein comprises an antibody that binds human EpCAM and that comprisesan antibody light chain comprising an amino acid sequence defined byresidues 1-106 of SEQ ID NO:9 and an antibody heavy chain comprising anamino acid sequence defined by residues 1-116 of SEQ ID NO:18.
 32. Thenucleic acid of claim 31, wherein the protein comprises a cytokine. 33.The nucleic acid of claim 32, wherein the cytokine is IL-2.
 34. A cellcomprising the nucleic acid of claim
 31. 35. A nucleic acid encoding ahumanized or deimmunized anti-EpCAM protein suitable for administeringinto a human patient for treating a disease associated with EpCAMover-expression, wherein the protein comprises an antibody that bindshuman EpCAM and that comprises: (a) an amino acid sequence selected fromthe group consisting of: (i) amino acid residues 24-31 of SEQ ID NO: 1;(ii) amino acid residues 49-55 of SEQ ID NO: 1; and (iii) amino acidresidues 88-96 of SEQ ID NO: 1; the antibody further comprising (b) anamino acid sequence defining an immunoglobulin light chain frameworkregion selected from the group consisting of: (i) amino acid residues1-23 of SEQ ID NO:8; and (ii) amino acid residues 1-23 of SEQ ID NO:9.36. A nucleic acid encoding a humanized or deimmunized anti-EpCAMprotein suitable for administering into a human patient for treating adisease associated with EpCAM over-expression, wherein the proteincomprises an antibody that binds human EpCAM and that comprises: (a) anamino acid sequence selected from the group consisting of: (i) aminoacid residues 26-35 of SEQ ID NO:2; (ii) amino acid residues 50-62 ofSEQ ID NO:2; and (iii) amino acid residues 101-105 of SEQ ID NO:2; theantibody further comprising (b) an amino acid sequence defining animmunoglobulin heavy chain framework region selected from the groupconsisting of: (i) amino acid residues 1-25 of SEQ ID NO: 18; and (ii)amino acid residues 67-98 of SEQ ID NO:18.
 37. A nucleic acid encoding ahumanized or deimmunized anti-EpCAM protein suitable for administeringinto a human patient for treating a disease associated with EpCAMover-expression, wherein the protein comprises an antibody that bindshuman EpCAM and that comprises an antibody light chain comprising anamino acid sequence defined by residues 1-106 of SEQ ID NO:9 and anantibody heavy chain comprising an amino acid sequence defined byresidues 1-116 of SEQ ID NO:18.